MiR-146a 在长波紫外线诱导人皮肤成纤维细胞光老化中作用机制的研究

Role of miR-146a in ultraviolet A-induced photoaging of human skin fibroblasts and its mechanism

目的：探讨长波紫外线（UVA）诱导人皮肤成纤维细胞（HSF）光老化中 miR-146a 的表达情况，以及上调 miR-146a 表达对其靶基因 Smad4及细胞光老化的影响。方法以10 J/cm2 UVA 照射 HSF （UVA 照射组），分别在0、3、7、14 d 提取 RNA，实时定量 PCR 检测 miR-146a 的表达量。通过慢病毒转染上调 miR-146a 的表达（miR-146a 过表达组），在7 d、14 d 后用荧光显微镜观察转染效率，并通过实时定量 PCR 验证细胞内miR-146a 表达量。空白对照组为正常培养的 HSF（不做任何处理），miR-146a 过表达组为慢病毒转染 HSF 后，再用 UVA 照射。MTT 法检测空白对照组、UVA 照射组、miR-146a 过表达组、miR-146a 过表达组细胞增殖吸光度（A 值），实时定量 PCR 检测各组细胞内老化相关基因 p53、p16和 p21 mRNA 的表达，Western 印迹法检测各组细胞内 Smad4蛋白表达。采用重复测量的方差分析、析因设计的方差分析进行统计学分析。结果重复测量的方差分析显示，随培养时间的延长，UVA 照射组和空白对照组 miR-146a 的表达量均逐渐下降（F =213.840， P 〈0.01）；UVA 照射组表达量低于空白对照组（F =52.55，P 〈0.01），且照射时间越长，下调越明显。慢病毒转染HSF 后，细胞内均有较高的荧光表达，miR-146a 过表达组在第7天（10.31±0.17）与第14天时（9.65±0.19）的miR-146a 表达量差异无统计学意义（P 〉0.05），但较空白对照组（分别为8.33±0.13和7.86±0.11）显著增高，组间差异有统计学意义（F =42.49，P 〈0.01）。析因设计的方差分析显示，UVA 照射对细胞增殖活性有抑制作用（P 〈0.01），UVA 照射组、UVA ＋ miR-146a 组细胞增殖均分别低于空白对照组、miR-146a 过表达组（P 〈0.01）；慢病毒转染上调 miR-146a 的表达对细胞增殖活性也有影响（P 〈0.01），但 miR-146a 过表达组与空白对照组差异无统计学意义（P 〉0.05），UVA ＋ miR-146a 组显著高于 UVA 照射组（P 〈0.01）。实时定量 PCR 和 Western 印迹法结果显示，UVA 照射可上调 p21、p53、p16 mRNA 的表达（均 P 〈0.01），同时对细胞内 Smad4蛋白表达有促进作用（P 〈0.01）；UVA 照射组、UVA ＋ miR-146a 组 p21、p53、p16 mRNA 和 Smad4蛋白表达均分别高于空白对照组、miR-146a 过表达组（均 P 〈0.01），而 miR-146a 过表达组与空白对照组相比差异均无统计学意义（均 P 〉0.05）， UVA ＋ miR-146a 组均显著低于 UVA 照射组（均 P 〈0.01）。结论在 UVA 诱导光老化的 HSF 中，miR-146a 的表达受到抑制，上调其表达能够抑制 Smad4的表达，促进光老化细胞增殖，起到抗细胞光老化的作用。

Objective To investigate miR-146a-Smad4 expression during ultraviolet A（UVA）-induced photoaging of human skin fibroblasts （HSFs）, and to evaluate effects of up-regulation of miR-146a expression on its target gene Smad4 and cell photoaging. Methods HSFs were isolated from the prepuce, and subjected to primary culture and maintained up to 10th passage. Then, the HSFs were classified into 4 groups： blank control group receiving no treatment, UVA group irradiated with 10 J/cm2 UVA, miR-146a group transfected with a lentiviral vector expressing miR-146a, UVA＋ miR-146a group transfected with the lentiviral vector expressing miR-146a followed by UVA radiation. Real time PCR was performed to measure miR-146a expression in HSFs in the UVA group on day 0, 3, 7 and 14 after UVA radiation.Fluorescence microscopy was carried out to estimate transfection efficiency on day 7 and 14 in the miR-146a group after transfection, and real time PCR was performed to quantify miR-146a expression in these cells. Methyl thiazolyl tetrazolium （MTT）assay was conducted to evaluate proliferative activity of HSFs, real time PCR to quantify mRNA expressions of photoaging-related genes p53, p21 and p16, and Western blot analysis to measure Smad4 protein expression in these cells. Statistical analysis was carried out by using repeated-measures analysis of variance and factorial design analysis of variance. Results Repeated-measures analysis of variance showed that the expression of miR-146a decreased over time in both the UVA group and blank control group（F = 213.840, P 〈 0.01）, and significantly lower in the UVA group than in the blank control group （F = 52.55, P 〈 0.01）, with the difference between the two groups increasing over time. After transfection with the lentiviral vector expressing miR-146a-Smad4, HSFs showed a strong fluorescence intensity of miR-146a. The expression level of miR-146a was significantly higher in the miR-146a group than in the blank control group on day 7 and 14 after transfection（10.31 ± 0.17 vs. 8.33 ± 0.13 on day 7, 9.65 ± 0.19 vs. 7.86 ± 0.11 on day 14, F =42.49, P 〈 0.01）, but insignificantly different between day 7 and 14 in the miR-146a group （P 〉 0.05）. Factorial design analysis of variance showed that UVA radiation had an inhibitory effect on the proliferative activity of HSFs （P 〈 0.01）, which was significantly lower in the UVA group than in the blank control group（P 〈 0.01）, and lower in the UVA ＋ miR-146a group than in the miR-146a group （P 〈 0.01）. The lentivirus-mediated up-regulation of miR-146a expression also affected cellular proliferative activity （P 〈 0.01）, which was significantly higher in the UVA ＋ miR-146a group than in the UVA group（P 〈 0.01）, but insignificantly different between the miR-146a group and blank control group（P 〉 0.05）. Real time PCR and Western blot analysis both revealed that UVA radiation could increase the expressions of p53, p21 and p16 mRNAs as well as Smad4 protein（all P 〈 0.01）. Concretely speaking, the expressions of p53, p21, p16 mRNAs and Smad4 protein were all significantly higher in the UVA group than in the blank control group （all P 〈 0.01）, and higher in the UVA ＋miR-146a group than in the miR-146a group （all P 〈 0.01）, but significantly lower in the UVA ＋ miR-146a group than in the UVA group （all P 〈 0.01）, and insignificantly different between the blank control group and miR-146a group （all P 〉0.05）. Conclusion The expression of miR-146a is inhibited in UVA-induced photoaged HSFs, and its up-regulation may counteract cell photoaging by suppressing Smad4 expression in, and promoting proliferation of, photoaged HSFs.